A reversibly glycosylated polypeptide (RGP1) possibly involved in plant cell wall synthesis: Purification, gene cloning, and trans-Golgi localization

Abstract

We purified from pea (Pisum sativum) tissue an Ϸ40 kDa reversibly glycosylated polypeptide (RGP1) that can be glycosylated by UDP-Glc, UDP-Xyl, or UDP-Gal, and isolated a cDNA encoding it, apparently derived from a single-copy gene (Rgp1). Its predicted translation product has 364 aminoacyl residues and molecular mass of 41.5 kDa. RGP1 appears to be a membrane-peripheral protein. Immunogold labeling localizes it specifically to trans-Golgi dictyosomal cisternae. Along with other evidence, this suggests that RGP1 is involved in synthesis of xyloglucan and possibly other hemicelluloses. Corn (Zea mays) contains a biochemically similar and structurally homologous RGP1, which has been thought (it now seems mistakenly) to function in starch synthesis. The expressed sequence database also reveals close homologs of pea Rgp1 in Arabidopsis and rice (Oryza sativa). Rice possesses, in addition, a distinct but homologous sequence (Rgp2). RGP1 provides a polypeptide marker for Golgi membranes that should be useful in plant membrane studies.

Sugar-nucleotide-binding and autoglycosylating polypeptide(s) from nasturtium fruit: biochemical capacities and potential functions

“…In all of these respects, the ["%C]Gal-labelled polypeptide from nasturtium resembled RGPs from pea and Arabidopsis. One difference in the results of pulse-chase reactions in the present tests versus earlier experiments was that UDP-Man from various suppliers was as effective as the UDP-sugars, which are precursors for XG at displacing UDP-["%C]Gal from nasturtium polypeptide (Table 2), although UDP-Man was ineffective at chasing UDP-["%C]Glc from pea and Arabidopsis polypeptide [1][2][3]. Another difference is that with nasturtium enzyme, UDPuronic acids were partially effective chase reagents, although UDP-N-acetyl sugar amines were not (Table 2).…”

“…The addition of β-mercaptoethanol resulted in the disappearance of many of the largest and smallest stained bands, with most of the polypeptides now clustered in bands between 40 and 65 kDa. The label was confined to one band at 41 kDa, which is the molecular mass reported for RGPs from peas [1,2], Arabidopsis [3] and mung bean [4].…”

“…These antibodies react with polypeptides of 41 and 66 kDa in extracts of Arabidopsis, pea, tobacco and maize [3]. In nasturtium profiles, anti-AtRGP1 recognized under reducing conditions a polypeptide with a molecular mass of 41 kDa, which is the size of the ["%C]Gal-labelled polypeptide (Figure 1) and the ["%C]Glc-labelled polypeptides from other plant sources [1][2][3][4]. It also reacted with a family of at least three nasturtium polypeptides with molecular masses between 62 and 70 kDa, which correspond to several of the bands that stain heavily with Coomassie Brilliant Blue but are not labelled with ["%C]Gal (Figure 1).…”

“…Polypeptides that become labelled upon incubation with UDP-["%C]Glc have been reported recently in membrane and soluble enzyme preparations from pea epicotyl [1,2], Arabidopsis, maize and tobacco [3]. The products fractionate after electrophoresis as a single band with a molecular mass of 41 kDa, and cDNA analysis shows substantial sequence homology between them.…”

“…A potential relationship as an intermediate or precursor for XG biosynthesis in pea [1,2] and Arabidopsis [3] was surmised from the facts that UDP-[$H]Xyl and UDP-[$H]Gal could chase ["%C]Glc from association with their polypeptides, and ["%C]Glc was replaced by the tritiated sugars. Immunogold labelling with antibody raised to Arabidopsis RGP localized it to trans-Golgi dictyosomal cisternae, where XG biosynthesis occurs [5,6], but not to plastids, plasma membrane or endoplasmic reticulum.…”